Structural basis for recognition of the Hsp90 closed, ATP state by the TPR‐containing co‐chaperone FKBP51

Abstract

The Hsp90 molecular chaperone drives activation of hundreds of client proteins in the cell through an ATP-dependent conformational cycle guided by distinct co-chaperone regulators. The FKBP51 immunophilin co-chaperone binds Hsp90 through its tetratricopeptide repeat (TPR) domain and catalyzes peptidyl-prolyl isomerase (PPIase) activity during the folding of kinases, nuclear receptors and tau. However, for many TPR cochaperones, the protein:protein interactions and mechanisms which enable Hsp90 specificity and regulation in the folding pathway remain unclear. High resolution structure of the Hsp90:FKBP51:p23 complex was determined using cryo-electron microscopy (EM). Native gel electrophoresis, gel filtration chromatography coupled with multiangle light scattering, and negative stain-EM were used to test interaction of FKBP51/p23 with Hsp90 in different nucleotide binding states. The structure of the Hsp90:FKBP51:p23 complex was verified with mutational analysis and site-specific photocrosslinking experiments. We developed in vitro conditions that promote and stabilize the closed, ATP conformation of Hsp90, enabling characterization of binding interactions between defined open (apo) and closed (ATP) states. We find that FKBP51 preferentially binds the closed, ATP state, forming a complex with a 2:1 stoichiometry that is further stabilized by p23 binding. Using cryo-EM we determined a 3.3 Å structure the Hsp90:FKBP51:p23 complex that reveals distinct contacts by the TPR domain of FKBP51 that define its asymmetric interaction and recognition of C-terminal Hsp90 dimer. We propose a model in which helix 7 of the TPR domain of FKBP51 functions as a critical specificity element for engaging Hsp90 in the p23-stabilized, ATP state to promote client maturation. This arrangement flexibly positions the FK1 domain of FKBP51 adjacent Hsp90 client binding sites in a manner we predict facilitates site-specific PPIase activity towards clients in coordination with the Hsp90 chaperone cycle.